Isolation, characterization and baculovirus-mediated expression of the cDNA encoding cytosine DNA methyltransferase from Pisum sativum.
A series of overlapping clones complementary to the Arabidopsis cytosine-5 DNA methyltransferase (C-5 MTase) has been isolated from pea cDNA libraries. The assembled nucleic acid sequence contains an open reading frame of 4761 bp encoding a protein of 1554 amino acids. Like other eukaryotic C-5 MTases, the inferred protein has a presumed regulatory N-terminal region linked to a catalytic C-terminal domain, which has eight of the ten conserved motifs found in prokaryotic C-5 MTases. The pea C-5 MTase has 65% identity at the nucleotide level and 61% identity at the protein level, with the Arabidopsis C-5 MTase. The catalytic domain of the pea enzyme shares 78% identity with Arabidopsis and approximately 52% identity with murine and human C-5 MTases, including the relative position of the proline-cysteine dipeptides of the catalytic centre. Using the conserved region of the cDNA as a probe, we have identified a transcript of 5 kb. Southern blot analysis of pea genomic DNA with the above probe indicates the presence of a single gene. Using poly(A)+ RNA from different developmental stages and different tissues, we have observed that expression is confined mostly to the rapidly dividing tissues of the plant. Expression of this assembled cDNA in a baculovirus system gives a protein of approximately 174 kDa. The expressed protein can be cross-linked, in an AdoMet-dependent manner, to duplex oligonucleotide substrates containing FdC in place of target cytosines in either CG or CAG/CTG sequences.
Pradhan
S
S
Division of Biochemistry and Molecular Biology, Institute of Biomedical and Life Sciences, Davidson Building, University of Glasgow, Glasgow G12 8QQ, UK.
Cummings
M
M
Roberts
R J
RJ
Adams
R L
RL
D000595:Amino Acid Sequence; D000818:Animals; D017360:Arabidopsis / Q000201:enzymology / Q000235:genetics; D016367:Baculoviridae / Q000235:genetics; D001483:Base Sequence; D003001:Cloning, Molecular; D017124:Conserved Sequence; D004248:DNA (Cytosine-5-)-Methyltransferases / Q000235:genetics*; D017931:DNA Primers / Q000235:genetics; D018076:DNA, Complementary / Q000235:genetics*; D018744:DNA, Plant / Q000235:genetics*; D015870:Gene Expression; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D018532:Pisum sativum / Q000201:enzymology* / Q000235:genetics*; D012333:RNA, Messenger / Q000235:genetics; D018749:RNA, Plant / Q000235:genetics; D017386:Sequence Homology, Amino Acid
D017931:DNA Primers; D018076:DNA, Complementary; D018744:DNA, Plant; D012333:RNA, Messenger; D018749:RNA, Plant; D004248:DNA (Cytosine-5-)-Methyltransferases